Western DotBlot protocol?

Ted Michelini tedm at darkwing.uoregon.edu
Mon Apr 24 23:02:31 EST 1995


In article <blume-2404952113370001 at mozarttty12.jvnc.net>,
blume at tigger.jvnc.net (John E. Blume) wrote:

> Does anyone have a favorite protocol for doing Western blots in some kind
> of dot/slot format? I have many samples to test for effect and I hope to
avoid  running a gel and transferring. Obviously, I am concerned about
background and about membrane binding capacity, but it strikes me that
someone must have this
> worked out.
> 
> Thanks,
> John Blume


John,
   The dot blot you refer to are usually done in a two fold dilution
series to give semi-quantitation, but they needn¹t be.  Similar dot blots
are routinely used to follow column elutions etc.
My experience entailed a direct application of yeast supernatant with
recombinant protein to an S&S square nitrocellulose membrane with a
scribed grid and intermediate pore size, a 1-2ul vol was about a 5mm dia
dot. Its possible to use a multipipettor if it is perfectly parallel and
fairly accurate at that low vol. After the spots have dried you block with
either a 5% BSA in PBS sol. or gelatin; NFDM may work as well.  This is
all done within a square petri plate, with gentle rotation and 3x PBS
washes between solutions. The primary antibody is then added in a solution
of the blocking buffer and rotated for ~30min; amounts are contingent on
previous titration. I reused the antibody usually 5-10 times with little
degradation, having included azide in all your solutions, of course. With
polyclonal antibodies it is important to preabsorb against other proteins
present; in my case native yeast supernatant. Choice of the pore size of
the nitrocellulose depends on your proteins size/behavior.
   The secondary antibody can be either an HRP  (or AP) or a
chemiluminescent system, the latters higher sensitivity being at the
expense of high background, often. The added benefit of the
chemiluminescent system is a more durable hard copy of the results and
possibly more reliable numbers from a scanning densitometer of the film. 
Reflectance from the nitrocellulose is not very accurate/sensitive. At
times the high background is remedied by a wash after the secondary
antibody with a 0.1% NP-40 solution. Good luck.
                                          Ted M.            my 0.02
                                          tedm at darkwing.uoregon.edu



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